Steinebach O M, Wolterbeek H T
Department of Radiochemistry, Delft University of Technology, The Netherlands.
Chem Biol Interact. 1992 Nov 16;84(3):199-220. doi: 10.1016/0009-2797(92)90124-4.
Experiments were carried out to investigate the uptake and accumulation of Zn in rat hepatoma HTC cells, as affected by interfering metals (Cd, Cu), metallothionein synthesis inhibiting compounds (Actinomycin D, cycloheximide) and metallothionein synthesis stimulating compounds (dexamethasone, dibu-cAMP). Cell viability was tested under all experimental conditions by the measurement of LDH leakage, K+ uptake and total cell protein. Determinations of Zn were performed by AAS (total Zn) or by gamma-ray spectrometry (65Zn). Metallothionein analysis was carried out by Cd-saturation tests. The results indicate that cellular responses in rat hepatoma HTC cells with respect to the uptake and accumulation of 65Zn are fully comparable with literature data existing for 65Zn accumulation in rat hepatocytes, under all experimental conditions applied. Cu2+ and dibutyryl-cAMP did not significantly affect rates of 65Zn accumulation. Cd2+, Actinomycin D and cycloheximide reduced 65Zn uptake, but dexamethasone additions resulted in increased 65Zn accumulation in the cells. Effects on 65Zn were shown both in cytosolic and in the membranes/organelles cell fractions. HPLC chromatography in control cells suggested that newly accumulated cytosolic 65Zn was predominantly MT-associated. Dexamethasone-induced 65Zn accumulation could not be related to elevated cellular MT levels, nor were the total cytosolic Zn levels significantly affected. Non-specific attenuations in MT levels (Actinomycin D, cycloheximide and dibu-cAMP) yielded linear relations between cytosolic 65Zn and MT levels, without any change in cytosolic Zn (AAS). Combined addition of Cd and dexamethasone yielded elevated MT levels, but severely reduced total cytosolic Zn and 65Zn concentrations. The results further indicate the non-Zn-specific nature of dexamethasone-action and suggest the relatively easy Zn-complexing and Zn-release of MT. The simultaneous determinations of total cytosolic zinc and cytosolic 65Zn levels showed that the application and sole measurement of radiotracers may yield only one-sided views of what is actually present or occurring in the cells.
开展实验以研究大鼠肝癌HTC细胞中锌的摄取和积累情况，该过程受干扰金属（镉、铜）、金属硫蛋白合成抑制化合物（放线菌素D、环己酰亚胺）和金属硫蛋白合成刺激化合物（地塞米松、二丁酰环磷腺苷）的影响。在所有实验条件下，通过测量乳酸脱氢酶泄漏、钾摄取和总细胞蛋白来检测细胞活力。锌的测定通过原子吸收光谱法（总锌）或γ射线光谱法（65锌）进行。金属硫蛋白分析通过镉饱和试验进行。结果表明，在所有应用的实验条件下，大鼠肝癌HTC细胞对65锌摄取和积累的细胞反应与大鼠肝细胞中65锌积累的现有文献数据完全可比。铜离子和二丁酰环磷腺苷对65锌积累速率没有显著影响。镉离子、放线菌素D和环己酰亚胺降低了65锌的摄取，但添加地塞米松导致细胞中65锌积累增加。对65锌的影响在细胞溶质以及细胞膜/细胞器细胞组分中均有体现。对照细胞中的高效液相色谱表明，新积累的细胞溶质65锌主要与金属硫蛋白相关。地塞米松诱导的65锌积累与细胞内金属硫蛋白水平升高无关，细胞溶质总锌水平也未受到显著影响。金属硫蛋白水平的非特异性降低（放线菌素D、环己酰亚胺和二丁酰环磷腺苷）导致细胞溶质65锌与金属硫蛋白水平之间呈线性关系，而细胞溶质锌（原子吸收光谱法）没有任何变化。镉和地塞米松联合添加导致金属硫蛋白水平升高，但细胞溶质总锌和65锌浓度严重降低。结果进一步表明地塞米松作用的非锌特异性性质，并表明金属硫蛋白相对容易与锌络合和释放锌。细胞溶质总锌和细胞溶质65锌水平的同时测定表明，放射性示踪剂的应用和单独测量可能仅产生关于细胞中实际存在或发生情况的片面观点。
